Atherosclerosis is the major causative factor of heart disease and stroke, and cardiovascular disease is the leading cause of death in Western countries. Dyslipidemia is a primary contributor to atherosclerosis. Because triglycerides are insoluble in the bloodstream, they are packaged for plasma transport into micelle-like lipoprotein particles composed of protein, phospholipid and cholesterol shells surrounding a non-polar core of acylglycerols, free cholesterol, and cholesterol esters. Lipoproteins have been classified into five broad categories on the basis of their functional and physical properties: chylomicrons (which transport dietary lipids from intestine to tissues); very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL and low density lipoproteins (LDL), (all of which transport triacylglycerols and cholesterol from the liver to tissues); and high density lipoproteins (HDL) (which transport endogenous cholesterol from tissues to the liver, as well as mediating selective cholesteryl ester delivery to steroidogenic tissues). All of these particles undergo continuous metabolic processing and have somewhat variable properties and compositions. Plasma concentrations of LDL and HDL are directly and inversely related, respectively, to the risk of atherosclerotic cardiovascular disease (Acton et al., Mol. Med. Today, 1999, 5, 518-524; Krieger, Proc. Natl. Acad. Sci. U. S. A., 1998, 95, 4077-4080).
A variety of integral membrane glycoproteins, collectively designated as scavenger receptors, mediate the binding and uptake of native and modified lipoproteins. A structurally diverse group, the scavenger receptors have been subclassified according to their composition: the class A scavenger receptors are trimeric glycoproteins whereas the class B scavenger receptors are composed of a single glycosylated polypeptide. The scavenger receptors are able to interact with a broad range of ligands, including modified proteins, lipoproteins and some polyanionic polysaccharides, as well as with in vitro oxidized and senescent cells, polyanionic phospholipids, and bacterial components (Calvo et al., J. Lipid Res., 1998, 39, 777-788).
LDL catabolism involves endocytosis and degradation of the entire lipoprotein particle by a well-characterized LDL receptor. In contrast, HDL particles deliver the cholesterol component to cells without the degradation of the protein component of HDL; this process is known as selective lipid uptake. Thus, HDL delivers cholesteryl ester to nonplacental steroidogenic tissues (ovary, adrenal gland, and testis) for hormone synthesis and transport cholesterol from extrahepatic tissues to the liver (reverse cholesterol transport), and the receptor that mediates HDL-binding and selective lipid uptake is the class B scavenger receptor, CD36 antigen-like 1 (CD36L1; also known as CD36L1; CLA-1; and scavenger receptor class B type 1; SRB1; and the mouse homologue, SR-BI) (Acton et al., Science, 1996, 271, 518-520).
A human CD36L1 cDNA representing a third member of a novel gene family that also includes CD36 (the thrombospondin and collagen type I receptor) and the lysosomal integrin membrane protein II (LIMPII) was identified and isolated from the melanoma cell lines 14Mel, SKMel131, and SKMel37 as well as from Burkitt lymphoma cell Ramos using a PCR-based approach. Subsequently, the full-length CD36L1 gene was cloned from a cDNA library derived from PMA-stimulated HLA60 cells. An alternative splice form of the CD36L1 mRNA was also identified, and Northern analysis revealed the presence of a 2.9-kilobase transcript in all cell types examined, but expression varied significantly with cell type, with low levels of CD36L1 expression in lymphoid-derived cells and higher levels of expression in melanoma cells (Calvo and Vega, J. Biol. Chem., 1993, 268, 18929-18935). The hamster CD36L1 gene was identified by expression cloning as a class B scavenger receptor which bound modified lipoproteins such as acetylated LDL and oxidized LDL as well as native LDL (Acton et al., J. Biol. Chem., 1994, 269, 21003-21009). Using a panel of human-hamster somatic cell hybrids, the human CD36L1 gene was mapped to chromosome 12 (Calvo et al., Genomics, 1995, 25, 100-106), later refined to the 12q24.2 chromosomal locus (Cao et al., J. Biol. Chem., 1997, 272, 33068-33076).
The uptake of lipoprotein-derived cholesteryl esters through the CD36L1 pathway represents a high-capacity, hormone-inducible cholesterol delivery system to cells. CD36L1 was observed to transfer more tree cholesterol than cholesteryl esters to cells from either LDL or HDL, and most of the cholesterol that entered cells via CD36L1 was available for efflux, suggesting that most of this cholesterol remained in the plasma membrane. While CD36L1 was able to mediate the selective uptake of core lipids from both classes of lipoproteins, HDL was a more efficient donor of cholesteryl esters than LDL. Sterols transported from LDL or HDL by CD36L1 were equally effective in delivering cholesterol to the intracellular regulatory cholesterol pool, resulting in important regulatory effects on SREBP-2 (sterol regulatory element binding protein-2) and HMG-COA reductase, two genes important in cholesterol metabolism. Thus, CD36L1 is equally efficient at mediating the import and export of cholesterol to and from cells to lipoproteins and other acceptors, resulting in a rapid exchange of cholesterol between lipoproteins and cell membranes (Stangl et al., J. Biol. Chem., 1999, 274, 32692-32698).
The C-terminal linking and modulating protein (CLAMP) was identified in rat liver sinusoidal plasma membrane as a protein associated with the C-terminus of the CD36L1 protein. CLAMP may be involved in modulating the intracellular transport and metabolism of cholesteryl esters taken up from HDL by CD36L1 (Ikemoto et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 6538-6543).
The protective, antiatherogenic effect of HDL is believed to involve the reverse transport of cholesterol from cells in the arterial wall to the liver for disposal. HDL also reduces endotoxic activity of cholesterol by complexation and neutralization of lipopolysaccharide (LPS). CD36L1 expression is upregulated during phagocytic as well as dendritic differentiation of monocytes, indicating a role for this receptor in cholesterol homeostasis in phagocytes and antigen-presenting cells. Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that heterodimerize with the retinoid X receptor to act as ligand-activated transcriptional regulators of genes controlling lipid and glucose metabolism. CD36L1 is expressed in atherosclerotic lesion macrophages and is induced by PPAR activation, identifying a mechanism by which PPARs regulates cholesterol homeostasis via CD36L1 expression in atherosclerotic lesion macrophages (Chinetti et al., Circulation, 2000, 101, 2411-2417).
Conversely, CD36L1 expression is suppressed in monocytes and macrophages by proinflammatory stimuli such as LPS, the cytokine interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α). Expression of CD36L1 mRNA and protein was studied in an immortalized human aortic intima smooth-muscle cell line ISS10, and IFN-γ was found to inhibit CD36L1 protein, but not mRNA expression in smooth-muscle cells, indicating that CD36L1 could play an important role in atherogenesis and that IFN-γ might impact the progression of an atherosclerotic lesion (Imachi et al., Horm. Metab. Res., 2001, 33, 389-393). It has been hypothesized that suppression of CD36L1 expression during early inflammation decreases cholesterol efflux from monocytes/macrophages and thereby reduces the clearance of HDL particles from the circulation, helping to maintain the lipoprotein status in the serum important for neutralizing LPS in the circulation as a short-term means of host defense (Buechler et al., Biochem. Biophys. Res. Commun., 1999, 262, 251-254)
The specific recognition of anionic phospholipids in the outer leaflets of cell membranes and lipoproteins by cell surface receptors it believed to play an important role in physiologic and pathophysiologic processes such as recognition of damaged or senescent cells by the reticuloendothelial system and lipoprotein homeostasis. In addition to its role in the absorption of dietary lipids, CD36L1 can bind to the membrane phospholipid phosphatidylserine (PS) and phosphatidylinositol (PI)-containing liposomes with high affinity (Rigotti et al., The J. Biol. Chem., 1995, 270, 16221-16224). Chinese hamster ovary (CHO) cell lines constitutively expressing CD36L1 were found to recognize both negatively charged liposomes and apoptotic cells presumably bearing PS in the outer layer of the plasma membrane (Fukasawa et al., Exp. Cell Res., 1996, 222, 246-250). Furthermore, because expression of human CD36L1 was found in circulating monocytes and, to a lesser extent, in fully differentiated macrophages, and because apoptotic thymocytes were able to bind cells transfected with CD36L1, it appears that CD36L1 not only acts as a “docking receptor” for HDL in liver and steroidogenic tissues, but also has alternative functions in leukocytes as a means for recognition of damaged cells (Murao et al., J. Biol. Chem., 1997, 272, 17551-17557).
Aberrant cell proliferation is one of the hallmarks of carcinogenesis, and cholesterol is believed to be involved in cell proliferation and cancer progression. An association between high HDL and cholesterol and the incidence of breast cancer, possibly related to estrogen metabolism, has been reported. In a study of pathways that could contribute to enhanced proliferation rates of cancer cells, the human breast cancer HBL-100 cell line was able to acquire HDL-cholesteryl esters via selective uptake mediated by CD36L1. Subsequent hydrolysis by hormone-sensitive lipase provided a significant contribution to the free cholesterol pool in rapidly dividing HBL-100 cells, potentially reflecting a source of precursors for hormone synthesis and cancer cell proliferation (Pussinen et al., Biochem. J., 2000, 349, 559-566).
CD36L1 is also involved in synthesis of steroid hormones. Northern analyses of 42 tissue samples revealed expression of CD36L1 mRNA in normal adult and fetal adrenal tissues as well as in pathological tissues, such as hyperplasias and adrenocortical adenomas. Adrenocortical carcinomas and the adrenals adjacent to Cushing's adenomas expressed lower levels of CD36L1 mRNA than did normal tissues, and the accumulation of CD36L1 mRNA in primary cultures of normal adrenocortical cells was up-regulated by adrenocorticotropic hormone (ACTH) in a dose- and time-dependent manner through the cAMP-dependent protein kinase pathway, suggesting that CD36L1 is involved in selective cholesterol uptake in human adrenocortical cells (Liu et al., J. Clin. Endocrinol. Metab., 1997, 82, 2522-2527). The SF-1 protein, an orphan member of the nuclear hormone receptor family of transcription factors, is expressed at high levels in steroidogenic tissues and appears to regulate CD36L1. SF-1 binds, in a sequence-specific manner, to the promoter of CD36L1, and efficient transcription from the CD36L1 promoter in Y1 mouse adrenocortical cells was found to depend on an intact SF-1 site, suggesting that CD36L1 supplies selected tissues with lipoprotein-derived lipids and is part of the repertoire of SF-1 responsive genes involved in steroidogenesis (Cao et al., J. Biol. Chem., 1997, 272, 33068-33076). In support of these findings, homozygous null CD36L1 knockout mice have been generated and used to show that CD36L1 is not only required for maintaining normal biliary cholesterol levels, but also for oocyte development and female fertility (Trigatti et al., Proc. Natl. Acad. Sci. U. S. A., 1999, 96, 9322-9327). Thus, inhibition of CD36L1 could act as a means of reducing production of steroid hormones for contraception.
In another study of the effects of CD36L1 in mice, the transgene-induced overexpression of murine CD36L1 resulted in a stimulation of excretion of cholesterol into the bile and suppressed percentage dietary cholesterol absorption. Thus, by extension, accelerated reverse cholesterol transport and storage in the liver, induced by hepatic overproduction of CD36L1 might be associated with increase bile cholesterol content, increasing the risk of biliary cholesterol and gall stone disease in humans (Sehayek et al., Proc. Natl. Acad. Sci. U. S. A., 1998, 95, 10194-10199).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of CD36L1 and to date, investigative strategies aimed at modulating CD36L1 function have involved the use of antisense oligonucleotides.
A phosphorothioate antisense oligonucleotide, 27 nucleotides in length, corresponding to the 5′ end of the CD36L1 mRNA was used to suppress CD36L1 protein expression by 80% and to inhibit the binding of thymic macrophages and nursing thymic epithelial cells to apoptotic thymocytes by 40% (Imachi et al., Lab. Invest., 2000, 80, 263-270).
Disclosed and claimed in U.S. Pat. No. 5,962,322 is the nucleic acid sequence for the hamster and murine CD36L1 genes and a method for selectively altering transport of lipid, cholesterol, lipoprotein or component thereof into and out of mammalian cells in an amount effective to alter plasma cholesterol comprising administering a composition in an amount effective to alter expression or activity of CD36L1 and thus alter the rate of clearance of the protein component of HDL as compared to the cholesterol ester component of the HDL, wherein transport of lipid, cholesterol, lipoprotein or component of the lipoprotein is inhibited or stimulated by administering a compound which binds to a regulatory nucleic acid sequence and therefore inhibits or increases expression of CD36L1, and wherein the compound is a viral vector encoding CD36L1. Direct inhibitors such as antisense oligonucleotides are generally disclosed (Kozarsky et al., 1999).
Disclosed and claimed in PCT publication WO 99/11288 is a method for modifying steroid production in a mammal comprising administering a compound altering the transfer of cholesterol or cholesteryl ester from high density lipoprotein or other lipoproteins via CD36L1 to liver or steroidogenic tissues, wherein the compound alters CD36L1 expression in the tissue, and wherein the compound alters binding of CD36L1 to high density lipoprotein including cholesteryl ester or other lipoproteins, or CD36L1 binding to lipoprotein or transfer of cholesteryl ester, and wherein the mammal is a female and the compound is administered in an amount effective to prevent normal reproductive function, and wherein the mammal has a disorder characterized by overproduction or underproduction of steroids, as well as a method of manufacture of said compound and a pharmaceutical composition for use in any one of said methods. Direct inhibitors such as antisense oligonucleotides are generally disclosed (Krieger, 1999).
Disclosed and claimed in U.S. Pat. No. 6,130,041 are isolated intronic and polymorphic nucleic acid variants of a genomic DNA comprising the human CD36L1 gene, a kit for amplifying and/or for determining the molecular structure of at least a portion of a CD36L1 gene, a method for determining whether a subject has, or is at risk of developing, a disease or condition associated with a specific allelic variant of a polymorphic region in the human CD36L1 gene, further comprising determining whether the CD36L1 gene of the subject comprises an allelic variant that is associated with a disease or condition, wherein the disease or condition is an abnormal lipid metabolism, inappropriate lipid levels, a cardiovascular disease, atherosclerosis, gallstone formation or an abnormal body mass index. Antisense techniques are generally disclosed (Acton, 2000).
Disclosed and claimed in U.S. Pat. No. 5,965,790 is an isolated nucleic acid molecule which is capable of hybridizing to a nucleic acid molecule consisting of the nucleotide sequence the human CD36L1 promoter or the complement thereof, wherein the nucleic acid is capable of modulating transcription of a gene operably linked to the nucleic acid, and wherein the gene encodes a CD36L1 receptor, and wherein the nucleic acid is capable of activating or enhancing transcription of said gene, as well as vectors, host cells, transgenic mice. Antisense oligonucleotides are generally disclosed (Acton, 1999).
The results of investigations of CD36L1 discussed herein suggest that pharmacological modulation of CD36L1 activity and/or expression may be an appropriate point for therapeutic intervention in pathologic conditions such as the overproduction of biliary cholesterol and gall stone disease, atherosclerosis and potentially, as a method of contraception. Consequently, there remains a long-felt need for therapeutic agents capable of inhibiting the function of CD36L1.
Antisense technology is emerging as an effective means of reducing the expression of specific gene products and may therefore serve as a unique strategy with which to modulate the expression of CD36L1.
The present invention provides compositions and methods for modulating CD36L1 expression.